Two fundamentally different aging phenomena have been described at the cellular level: 1) the gradual decline of life processes in postmitotic cells, and, 2) the decline and eventual complete cessation of cell division observed in most replicating cell lineages. The former is measured in simple chronological time, and comes into play during the aging of postmitotic adult organisms, such as the nematode, or during the aging of largely postmitotic tissues such as the brain or muscle in more complex organisms. In contrast, finite replicative lifespan, often referred to as cellular replicative senescence , is measured in terms of cell divisions rather than chronological time. The current consensus is that both postmitotic and replicative aging processes are causally related to the aging of humans. The topic of this research proposal is the molecular mechanism of replicative aging processes, specifically, the molecular machine that actually executes and maintains senescence. The major tool will be targeted homologous recombination (gene targeting), which will be performed in normal (nonimmortalized) human cell strains of both fibroblastic and epithelial origin. The targets of gene targeting will be the following genes: the tumor suppressors p53 and retinoblastoma (Rb), and the Cdk kinase inhibitors p16INK4A, p19ARF, and p21CIP1/WAF1. The objective will be to ablate gene action and subsequently investigate the resultant senescence phenotypes on both the cellular and molecular levels. This direct interventive approach is expected to reveal the functionally relevant components of the molecular senescence machine. The proposal is built on a model which predicts that the molecular machine that establishes senescence is composed of components that also play roles in cell cycle control during the normal proliferative lifespan of the cell. The experiments in this proposal are designed to test the hypothesis that three events are required to convert a normal human cell into an immortal cell: 1) loss of p53-p21 pathway function, 2) loss of pl6-Rb pathway function, 3) gain of telomerase function. Experiments will be performed in normal human cells grown in in vitro cell culture. This is because a large body of evidence indicates that the regulation of replicative senescence mechanisms is significantly different in humans and in rodents. Therefore, due to the ethical unacceptability of experimentally altering the human germ line, and the limited utility of the rodent model to address the specific issues under investigation, the whole-organism transgenic route is not appropriate and the experimental model has been confined to human somatic cell culture.